Silver Ion High-Performance Liquid Chromatography—Atmospheric Pressure Chemical Ionization Mass Spectrometry: A Tool for Analyzing Cuticular Hydrocarbons

Aliphatic hydrocarbons (HCs) are usually analyzed by gas chromatography (GC) or matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. However, analyzing long-chain HCs by GC is difficult because of their low volatility and the risk of decomposition at high temperatures. MALDI cannot distinguish between isomeric HCs. An alternative approach based on silver ion high-performance liquid chromatography (Ag-HPLC) is shown here. The separation of HC standards and cuticular HCs was accomplished using two ChromSpher Lipids columns connected in series. A gradient elution of the analytes was optimized using mobile phases prepared from hexane (or isooctane) and acetonitrile, 2-propanol, or toluene. HCs were detected by atmospheric pressure chemical ionization mass spectrometry (APCI-MS). Good separation of the analytes according to the number of double bonds, cis/trans geometry, and position of double bonds was achieved. The retention times increased with the number of double bonds, and trans isomers eluted ahead of cis isomers. The mobile phase significantly affected the mass spectra of HCs. Depending on the mobile phase composition, deprotonated molecules, molecular ions, protonated molecules, and various solvent-related adducts of HCs were observed. The optimized Ag-HPLC/APCI-MS was applied for characterizing cuticular HCs from a flesh fly, Neobellieria bullata, and cockroach, Periplaneta americana. The method made it possible to detect a significantly higher number of HCs than previously reported for GC or MALDI-MS. Unsaturated HCs were frequently detected as isomers differing by double-bond position(s). Minor HCs with trans double bonds were found beside the prevailing cis isomers. Ag-HPLC/APCI-MS has great potential to become a new tool in chemical ecology for studying cuticular HCs.


Introduction
Aliphatic hydrocarbons (HCs) are the main constituents of petroleum. They are also biosynthesized by eukaryotic [1][2][3][4] and prokaryotic [5] organisms. Aliphatic HCs are commonly found in the waxy cuticular layer on the surface of higher plants and insects. HCs in plant epicuticular waxes help to decrease surface wetting or water loss and protect plants from ultraviolet light [1]. Insect cuticular HCs are usually complex mixtures of nalkanes, mono-, di-, trimethyl alkanes, and unsaturated HCs with cis double bonds. Besides their main function in cuticle protection, the genetically encoded mixtures of cuticular HCs often serve as semiochemicals [2][3][4].

Ag-HPLC of Hydrocarbons
The HPLC conditions were optimized to achieve good separation of HCs. Since longchain unsaturated hydrocarbons were not commercially available, we used a mixture of cuticular HCs isolated from the flesh fly N. bullata. The composition of the fly cuticular HCs is known; the mixture contained aliphatic HCs with up to 46 carbons and 0-3 double bonds [7,23,54,55]. Insects produce unsaturated HCs mainly with cis double bonds, which would not allow us to optimize the method fully. Therefore, we also used a synthetic mixture of six monounsaturated HCs with cis and trans double bonds.
The Ag-HPLC separation system was optimized using ChromSpher Lipids columns and gradients elution with mobile phases composed of hexane/2-propanol/acetonitrile (solvent system I), isooctane/2-propanol/acetonitrile (solvent system II), and hexane/toluene (solvent system III), see Table 1. The mobile phase systems were designed concerning their suitability for Ag-HPLC and APCI-MS detection of HCs [43,56,57]. Toluene and acetonitrile were assumed to interact predominantly with the silver ions, thus reducing the interactions with the double bonds. In contrast, methanol, 2-propanol, and acetone decreased the strength of the interactions with the silanol groups and other polar moieties [58]. Therefore, the composition of the mobile phase can be adjusted to achieve a high resolution of HC species differing in the number, configuration, and position of double bonds. Figure 1 shows Ag-HPLC/APCI-MS chromatograms of N. bullata HCs recorded in all three mobile phase systems. As expected, HCs eluted according to the increasing number of double bonds. The solvent systems I and II provided a similar pattern of peaks, and the retention times for saturated and monounsaturated HC were almost the same. Unsaturated HCs with two and three double bonds were strongly retained in the mobile phase containing hexane (system I). In the solvent systems I and II, di-and triunsaturated HCs eluted as sharp peaks, with no separation of the molecular species within these groups. The solvent system III (hexane/toluene) provided higher retention times for unsaturated HCs. Importantly, chromatographic separation of the molecular species within each group of unsaturated HCs was observed. The retention behavior of HCs was consistent with the previous study dealing with Ag-HPLC of n-alkadienes [32]. Earlier reports showed that the column temperature strongly influences chromatographic resolution in Ag-HPLC [36,56,58]. Therefore, the HCs separation in all three solvent systems was probed at 15 • C, 25 • C, and 35 • C ( Table 2). The retention times of unsaturated HCs were reduced by increasing temperatures in all three solvent systems. Surprisingly, the effect of the temperature was the complete opposite compared to the ester lipids reported previously [36,56,58]. The temperature effects in Ag-HPLC are explained by the different temperature-dependent stabilities of the complexes formed between silver ions and analytes [58]. In addition to the interactions of the chain double-bond electrons, silver ion complexes can also be formed by the interactions of carbonyls in ester lipids. Therefore, we assume that the absence of an ester group is responsible for the observed temperature effect. The retention order in all solvent systems followed the number of double bonds. Unfortunately, the column temperature did not influence the chromatographic resolution of the compounds with the same number of double bonds too much (Supplementary Figure S1) the observed temperature effect. The retention order in all solvent systems followed the number of double bonds. Unfortunately, the column temperature did not influence the chromatographic resolution of the compounds with the same number of double bonds too much (Supplementary Figure S1). No separation of the compounds with the same number of double bonds was observed in solvent systems I and II. The best separation of HCs in solvent system III was observed at 15 °C.  The effect of the steepness of the mobile phase gradient was investigated in the next step. In the solvent systems I and II, changing the gradient steepness (a linear increase in B in the range 1.220-0.278%/min) had no major effect on the chromatographic separation: HCs with the same number of double bonds coeluted. As regards the solvent system III, the chromatographic resolution was enhanced at slower gradients. Figure 2 shows Ag-HPLC chromatograms of N. Bullata HCs recorded using various gradient slopes (a linear increase in B at 1.00, 0.75, 0.66, and 0.50%/min) at 15 °C. The best  The effect of the steepness of the mobile phase gradient was investigated in the next step. In the solvent systems I and II, changing the gradient steepness (a linear increase in B in the range 1.220-0.278%/min) had no major effect on the chromatographic separation: HCs with the same number of double bonds coeluted. As regards the solvent system III, the chromatographic resolution was enhanced at slower gradients. Figure 2 shows Ag-HPLC chromatograms of N. Bullata HCs recorded using various gradient slopes (a linear increase in B at 1.00, 0.75, 0.66, and 0.50%/min) at 15 • C. The best separation was observed for the slowest gradient; further lowering the gradient slope would not be convenient because of long retention times, peak broadening, and decreased sensitivity.
Molecules 2023, 28, x FOR PEER REVIEW 5 of 18 separation was observed for the slowest gradient; further lowering the gradient slope would not be convenient because of long retention times, peak broadening, and decreased sensitivity.   Figure 3a-c) indicate the separation of the aliphatic HCs according to the number of carbon atoms. The decrease in retention times with the number of carbon atoms can be rationalized by a normal phase separation mode, which partially occurs in Ag-HPLC systems. Similar behavior was previously observed for TGs, WEs, and FAMEs [35].  The decrease in retention times with the number of carbon atoms can be rationalized by a normal phase separation mode, which partially occurs in Ag-HPLC systems. Similar behavior was previously observed for TGs, WEs, and FAMEs [35]. Cuticular HCs biosynthesized from fatty acids mostly contain cis double bonds. To investigate the effect of double-bond geometry, a synthetic mixture containing cis and trans isomers was used. The mixture of 13c-C26:1, 13c-C32:1, 19c-C38:1, 13t-C26:1, 13t-C32:1, and 19t-C38:1 was separated in solvent system III. Two clusters of peaks appeared in the chromatograms (Figure 4), indicating the separation of the cis/trans isomers. Excellent separation of cis/trans isomers of various lipids in Ag-HPLC is well known; trans isomers elute faster because their interactions with silver ions are less efficient due to a larger steric hindrance [35]. We used NMR to elucidate double-bond geometry in two 16-C32:1 isomers isolated by preparative Ag-HPLC. The NMR spectra of the isomers were similar and confirmed the molecule's symmetry, resulting in half the number of NMR signals. The symmetry equivalence of olefinic protons made it impossible to use their mutual coupling to distinguish cis and trans isomers. Therefore, we used a difference in 13 C chemical shifts of the allylic carbon atoms. The faster isomer showed an allylic carbon at 32.60 ppm, while the slower one was at 27.20 ppm. The −5.4 ppm shift in the slower isomer can be explained by the steric upfield shift between two methylene groups in the cis isomer. At the same time, such an effect is absent in the faster isomer with a trans double-bond configuration (see Figure 5). In this way, we confirmed that the trans isomer eluted faster. The column temperature moderately affected the chromatographic separation of species within the trans and cis isomer groups; the best resolution was observed at 15 • C (cf. Figure 4a-c). Cuticular HCs biosynthesized from fatty acids mostly contain cis double bonds. T investigate the effect of double-bond geometry, a synthetic mixture containing cis and trans isomers was used. The mixture of 13c-C26:1, 13c-C32:1, 19c-C38:1, 13t-C26:1, 13t C32:1, and 19t-C38:1 was separated in solvent system III. Two clusters of peaks appeared in the chromatograms (Figure 4), indicating the separation of the cis/trans isomers Excellent separation of cis/trans isomers of various lipids in Ag-HPLC is well known; tran isomers elute faster because their interactions with silver ions are less efficient due to larger steric hindrance [35]. We used NMR to elucidate double-bond geometry in two 16 C32:1 isomers isolated by preparative Ag-HPLC. The NMR spectra of the isomers wer similar and confirmed the molecule's symmetry, resulting in half the number of NMR signals. The symmetry equivalence of olefinic protons made it impossible to use thei mutual coupling to distinguish cis and trans isomers. Therefore, we used a difference in 13 C chemical shifts of the allylic carbon atoms. The faster isomer showed an allylic carbon at 32.60 ppm, while the slower one was at 27.20 ppm. The −5.4 ppm shift in the slowe isomer can be explained by the steric upfield shift between two methylene groups in th cis isomer. At the same time, such an effect is absent in the faster isomer with a tran double-bond configuration (see Figure 5). In this way, we confirmed that the trans isome eluted faster. The column temperature moderately affected the chromatographi separation of species within the trans and cis isomer groups; the best resolution wa observed at 15 °C (cf. Figure 4a-c).    To conclude the chromatographic experiments, two ChromSphe series at 15 °C and a hexane/toluene gradient with a toluene increa provided the best separation conditions for unsaturated HCs. Compa systems tested (systems I and II), the optimized method showed supe of the retention times and required the shortest equilibration ti miscibility of toluene and hexane positively affected the method's ro of the retention times were 1.1% at the most for the HPLC runs made = 3) and up to 2.3% for measurements made within a month (n = 6).

The APCI-MS Detection of HCs Separated by HPLC
A range of ions can be observed in APCI spectra of hydrocarbon + H] + , [M − H] + , solvent-related adducts, and fragments. The molecul formed by electron transfer reactions with ionized nitrogen species, wh from strong Brønsted acids such as N2H + and H3O + create [M + H] + [5 [M − H] + from saturated hydrocarbons was recently rationalized by eli hydrogen from protonated molecules [50]. Since the proton transfer exothermic, fragment ions are formed. Solvent-related adducts are p To conclude the chromatographic experiments, two ChromSpher Lipids columns in series at 15 • C and a hexane/toluene gradient with a toluene increase rate of 0.5%/min provided the best separation conditions for unsaturated HCs. Compared to other solvent systems tested (systems I and II), the optimized method showed superior reproducibility of the retention times and required the shortest equilibration times. The excellent miscibility of toluene and hexane positively affected the method's robustness. The RSDs of the retention times were 1.1% at the most for the HPLC runs made on the same day (n = 3) and up to 2.3% for measurements made within a month (n = 6).  [50]. The formation of [M − H] + from saturated hydrocarbons was recently rationalized by eliminating molecular hydrogen from protonated molecules [50]. Since the proton transfer reactions are highly exothermic, fragment ions are formed. Solvent-related adducts are products of chemical ionization processes in the ion source, and their nature reflects the mobile phase composition.

The APCI-MS Detection of HCs Separated by HPLC
The effect of various solvents on the APCI spectra was exemplified for 13c-C32:1. The mass spectra of this compound recorded in the solvent systems I, II, and III at the corresponding retention times are shown in Figure 6. In the solvent system I (Figure 6a source fragments of the aliphatic chain were also observed for all solvent systems. The intensities of the fragments were up to 8% of the base peak. highest among the systems tested. In the next experiments, we recorded APCI sp 16c-C32:1 dissolved in mobile phases differing by their proportions of hexane and (Figure 7). In 100% hexane, 16c-C32:1 yielded mostly [M + 39] +, and the intensity H] + was rather small. The intensity of [M + H] + increased with the increasing conce of toluene in the mobile phase and reached it maximum at 40-60% toluene. A fur in the toluene concentration caused a decrease in the [M + H] + peak. Ion source fr of the aliphatic chain were also observed for all solvent systems. The intensitie fragments were up to 8% of the base peak.   The ions formed from HCs in APCI also depended on the number of double bonds in their molecules. The number of double bonds significantly affects the detection sensitivity in many neutral lipids [59,60]. APCI spectra taken across chromatographic peaks thus reflected a combined effect of mobile phase composition and hydrocarbon structure. Figure 8 shows spectra taken across the most abundant chromatographic peaks in the N. bullata sample. Coeluting saturated HCs (Figure 8a)   The ions formed from HCs in APCI also depended on the number of double bond in their molecules. The number of double bonds significantly affects the detectio sensitivity in many neutral lipids [59,60]. APCI spectra taken across chromatographi peaks thus reflected a combined effect of mobile phase composition and hydrocarbo structure. Figure 8 shows spectra taken across the most abundant chromatographic peak in the N. bullata sample. Coeluting saturated HCs (Figure 8a) provided abundant signal of [M − H] + , which is in agreement with previous observations [46,50,61]. In contrast to saturated species, unsaturated HCs were detected as protonate molecules [M + H] + (Figure 8b-d) [46,53]. In addition to the protonated molecules, di-an In contrast to saturated species, unsaturated HCs were detected as protonated molecules [M + H] + (Figure 8b-d) [46,53]. In addition to the protonated molecules, di-and triunsaturated HCs eluting at high concentrations of toluene provided abundant radical cations [M] +· and also adducts with protonated toluene [M + 93] + (Figure 8c,d). As evident from previous paragraphs, interpreting the APCI spectra of HCs is not always straightforward. However, the spectra give enough information for a reliable assignment of the molecular weight, number of carbons, and number of double bonds.

Cuticular HCs of N. bullata
The optimized Ag-HPLC/MS method made it possible to detect 136 cuticular HCs in N. bullata and characterize them by the number of carbons, double bonds, and double-bond geometry ( Table 3). The relative peak areas were used to estimate the proportions of HCs. It is important to note that the relative peak areas do not correctly reflect real abundances, mainly because of large differences between the response factors of analytes with different numbers of double bonds. As discussed previously [59,60], APCI-MS tends to provide a higher response for unsaturated lipids and thus underestimates saturated species. The relative peak areas are reasonable estimates within the groups of compounds with the same degree of unsaturation. The chain length also influences the detection sensitivity, but its effect is less important [59,62].  Saturated HCs eluted close to the column dead volume. Chromatograms reconstructed for [M − H] + ions showed a gradual decrease in the retention time with the number of carbon atoms, indicating a normal phase separation mechanism (Table 3). Nevertheless, retention times were similar; all saturated HCs eluted within a ca. 7-second interval. Saturated HCs contained 26-49 carbon atoms, with C30:0, C45:0, and C47:0 being the most abundant species. Monounsaturated HCs were better resolved; they contained 27-47 carbons, with C37:1, C39:1, C40:1, and C42:1 providing the highest signals. The cis-C38:1 and cis-40:1 were detected at two different retention times, likely because of different double-bond positions. Their cis double-bond geometry was deduced from their retention times closely matching synthetically prepared standards (cf. Figure 4). Many di-and triunsaturated HCs, including isomers found in several chromatographically separated peaks, were detected. Diunsaturated HCs contained 37-45 carbons and cis double bonds; C39:2, C41:2, and C42:2 were the most abundantly present (Figure 3). Interestingly, minor HC peaks with a trans double bond(s) were observed (trans-monounsaturated HCs at R t 21.3-22.7; trans-diunsaturated HCs at R t 50-55.5 min ( Figure S2) and transtriunsaturated HCs at R t 84.1-89.9 min). Because of their low abundance (below the threshold), they are not listed in Table 3. The overall ratio of HCs with cis and trans (at least one) double bonds estimated from the peak areas was ca 1000:1. HCs with trans double bonds are extremely rare in insect cuticular HCs [2][3][4].
The overall number of HCs detected in N. bullata by Ag-HPLC/APCI-MS (136) was much higher than previously reported for GC [7,54,55] or MALDI-TOF MS [23]. Two reasons for more successful detection by HPLC/MS can be proposed: (a) an efficient chromatographic separation of unsaturated HCs in HPLC provides good resolution for isomeric HCs, and (b) the detection of nonvolatile (or degradable) long-chain HCs is not amenable to GC. Earlier papers based on GC reported 32 saturated HCs with 25-32 carbons, both straight-chain and branched [54,55]. The presence of heavier HCs was evident, but their characterization has not been performed for their low concentration. Later, HTGC/MS made it possible to detect almost 50 chromatographically separated peaks representing C25-C46 HCs. The nonpolar GC column provided limited selectivity for unsaturated HC isomers. It can be demonstrated by two isomers of C41:2 detected by HTGC/MS [7] and seven isomers of the same HCs resolved by Ag-HPLC (Table 3). MALDI-MS spectrum of HCs from N. bullata [7,23] showed ca 60 compounds with 25-49 carbons and 0-3 double bonds. The relatively low number of detected compounds resulted from detecting isobaric isomers at the same m/z values. On the other hand, summing up signals of very low abundant isomers with the same mass made it possible to detect them by MALDI-MS. This is probably why Ag-HPLC/APCI-MS detected unsaturated HCs up to C47, but MALDI-MS saw a little bit further and additionally detected C48 and C49 HCs. Hydrocarbons differing by one double bond can be difficult to distinguish in a MALDI spectrum because the signal of the more saturated hydrocarbon overlaps with the second isotopic peak of the less saturated specie.

Cuticular HCs of P. americana
Compared to N. bullata, the cuticular HCs from the cockroach P. americana were less complex. Ag-HPLC/APCI-MS disclosed 44 hydrocarbons (Table 4), 31 of which were saturated, 7 monounsaturated, 3 diunsaturated, and 3 triunsaturated. Saturated and monounsaturated species were eluted in narrow chromatographic zones; the signal of diunsaturated HCs was rather broad, which is likely due to a high abundance of C27:2 ( Figures S3 and S4 The monounsaturated HCs consisted of C27:1, C40:1, C41:1, C43:1, and C45:1, whereas C41:1 and C43:1 formed 91% of all monounsaturated HCs. Three different hydrocarbons corresponding to 27:1 were found. Diunsaturated HCs contained 27-29 carbons, and the major HC in this group was cis/cis-C27:2. As in the case of N. bullata, traces of HCs with one or two trans double bonds were detected at lower retention times ( Figure S5). Finally, the presence of triunsaturated HCs C41:3, C43:3, and C45:3 was observed at R t 117-119 min. As for N. bullata, Ag-HPLC/APCI-MS made it possible to detect significantly more cuticular HCs than the other methods. Existing literature based on GC reports 10 [63], 11 [64], or 27 [65] cuticular HCs in P. americana, which is about half of the HCs described in this work. MALDI MS made it possible to detect approximately 17 peaks of HCs [23]. In all reports, including this one, C27:2 was the major component of the cockroach's cuticular HCs.   [21]. The TLC spots were visualized by spraying rhodamine 6G solution (0.05% in ethanol), which was then scraped off the plates and extracted with diethyl ether. HCs were obtained from the upper part of the TLC plates (R F 0.5-1.0). Diethyl ether was removed under a stream of argon. Cuticular HCs were dissolved in chloroform to a concentration of 50 mg/mL (stock solution) and diluted before the analysis in chloroform to 10 mg/mL.

HPLC/APCI-MS
The liquid chromatograph consisted of an Accela autosampler, a Rheos Allegro UHPLC pump, and an LCQ Fleet ion-trap mass spectrometer. The system was controlled by Xcalibur software (all by Thermo Fisher Scientific, San Jose, CA, USA). Two Ag-HPLC ChromSpher Lipids columns (250 × 4.6 mm, particle size: 5 µm; Varian, Palo Alto, CA, USA) were connected in series. A column thermostat, which is a part of the autosampler, maintained the column temperature at 15 • C, 25 • C, or 35 • C. Three mobile phase systems were tested (Table 1); the gradient program started with 100% of solvent A, and the proportion of solvent B increased linearly during analysis. The flow rate of the mobile phase was 1.0 mL/min, and the injected volume of the samples was 3 µL or 5 µL. The columns were conditioned with 100% of A (100 µL/min) for at least 12 h before the first analysis and equilibrated for 1 h (solvent systems I and II) or 30 min (solvent system III) between injections. The APCI vaporizer and heated capillary temperatures were set to 350 • C and 150 • C, respectively; the corona discharge current was 3.

NMR
The NMR spectra were acquired with spectrometer Bruker AVANCE 500 ( 1 H at 500.1 MHz and 13 C at 125.8 MHz) that was equipped with a cryoprobe in CDCl 3 at 25 • C. Homonuclear 2D-NMR spectra (H,H-COSY) and heteronuclear 2D-NMR spectra (H,C-HSQC) were used for a partial structural assignment of proton and carbon signals and determination of double-bond configuration in olefinic isomers.

Conclusions
A new method for analyzing cuticular HCs was developed. Chromatographic separation according to the number and geometry of double bonds was achieved on an LC column containing silver ions. The mobile phase composition and gradient program were optimized, and the best results were achieved in a solvent system consisting of hexane and toluene. The optimized HPLC conditions made it possible to separate groups of saturated, mono-, di-, and triunsaturated HCs. Within the groups, a partial separation according to the chain length occurred. The chromatographic resolution increased with the number of double bonds. The great advantage of this method is the excellent separation of geometrical isomers, which allows cis and trans isomers to be resolved. Despite the formation of various structure-and solvent-dependent ions, APCI-MS detection made it possible to determine the molecular weight and, thus, the number of carbon atoms and double bonds. Ag-HPLC/APCI-MS efficiently detected long-chain HCs, which are difficult (or impossible) to analyze by GC/MS. Compared to MALDI-TOF, the main advantage of Ag-HPLC/APCI-MS is the ability to distinguish isomers and separate species differing by two hydrogens, i.e., a double bond. The increased sensitivity to HCs with one or more double bonds is another advantage of the method.
Further research is needed to find out how tandem mass spectrometry can be used to obtain more detailed information about the HCs structures (e.g., branching sites and positions of double bonds). Ag-HPLC/APCI-MS has great potential to become a new tool for studying cuticular HCs in chemical ecology. The method can discover new HCs that mediate various insect behaviors and cannot be detected by the existing analytical tools.

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Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.